Design
Overview
Our project aims to optimize the production of chitooligosaccharides (COS) by enzymatic degradation of chitin. First, chitin deacetylase (CDA) is used to convert chitin into chitosan, and then chitosanase (CsnB) hydrolyzes chitosan into COS. We constructed recombinant expression plasmids PET-28a-CDA and PET-28a-CsnB and connected the gene fragments to the vector using the Gibson assembly method to achieve enzyme expression. Additionally, we investigate mutations in CsnB to analyze enzymatic activity and the resulting products. We also compare dual enzyme synergistic catalysis and step-by-step catalysis in COS synthesis and explore the optimal catalytic conditions.
Project Goal
The degradation of chitin to produce chitosan is also known as enzymatic preparation of chitosan, i.e., as shown in Fig. 1. Chitin is firstly converted to chitosan under the action of chitin deacetylase, and chitosan is then converted to oligosaccharide by chitinase. The enzymatic degradation method differs from other degradation methods due to its mild reaction conditions, ease of process control, narrow molecular weight distribution of degradation products, less environmental pollution, and absence of side reactions. Therefore, our project focuses on modifying the enzyme (CsnB) used in the enzymatic preparation of COS to optimize the process for producing more high-purity COS.
Figure1 Chitin to chitosan reaction schematic[1, 2]
1. Recombinant Expression Vector Design
1.1 Chitin Deacetylase (CDA)
To realize the enzymatic hydrolysis process of chitin to chitosan, we constructed the expression plasmid of chitosan deacetylase (CDA). First, PET-28a was selected as the expression vector. Then, the CDA gene was extracted from the genome of Bacillus pumilus, and primers CDA-F and CDA-R were designed to specifically amplify the CDA gene fragment. Subsequently, the amplified CDA gene fragment was connected to the linearized PET-28a vector using the Gibson assembly method to form a recombinant plasmid PET-28a-CDA (Fig. 2).
Figure 2 Construction of PET-28a-CDA recombinant plasmid and gene circuit design of CDA expression.
1.2 Chitosanase (CsnB)
The chitosanase CsnB gene was derived from strain Marine Bacterium Bacilius SP. BY01.CsnB-F and CsnB-R were used as primers to amplify the CsnB gene fragment. Gibson assembly was used to ligate the CsnB gene fragment to the PET-28a linearized vector to obtain PET-28a-CsnB (Fig. 4).
Figure 4 Construction and gene circuit design of PET-28a-CsnB recombinant plasmid
2. Targeted Mutation of CsnB
In order to obtain a chitosanase that can hydrolyze and produce oligosaccharides with a single degree of polymerization, we will use a series of modeling techniques to predict protein structure and analyze the interactions between protein and small molecules. After that, we will perform site-directed mutagenesis on the amino acid sequence to obtain the mutant variants.
The chitosanase gene CsnB of Bacilius sp. BY01 was looked up from the NCBI database, and the structure of CsnB was predicted as follows using AlphaFold3 for modeling. Through reading the literature and related knowledge[7-13], we designed the following mutations: Val186 to Tyr (V186Y), Asp78 to Tyr's (D78Y), Lys260 to Trp's (K260W), Pro115 to Ala's (P115A). Prior to mutation we performed docking simulations between mutation sites and substrates using Autodock Vina and structure visualization analysis using PyMOL.
CsnB mutant vector design
We chose E. coli as the host for protein expression. PET-28a plasmid was used as the plasmid backbone to construct CsnB mutants’ repression vectors (Fig 5). The expression plasmid contains core components such as T7 promoter, lac operator, CsnB mutant gene, 6×His tag, kanamycin antibiotic gene, and T7 terminator. By inducing efficient expression of the CsnB mutant protein through the addition of IPTG, the protein can subsequently be purified using the 6×His tag for further enzyme activity tests and product polymerization.
Figure 5 pET-28a-CsnBD78Y, pET-28a-CsnBP115A, pET-28a-CsnBVB6Y, pET-28a-CsnBK260Y recombinant plasmid
3. Enzyme Performance Test
3.1 CDA Enzyme Performance
CDA Enzyme Activity Assay
The enzyme activity of CDA was performed by chromogenic substrate method which is a common way to quantify amidase activity. We selected p-nitroacetanilide as the substrate, which can be hydrolyzed to p-nitroaniline (Fig. 6). The concentration of p-nitroaniline can be measured by the characteristic absorption at 400 nm.
Figure 6 p-nitroaniline
Chitin deacetylase (CDA) activity unit is defined as the amount of enzyme required to produce 1 μg of p-nitroaniline per hour is defined as one unit of enzyme activity.
Then we obtained CDA Enzyme Activity by using the enzyme activity formula (1):
Formula 1
- A400: OD400 of experiment groups,
- A0: OD400 of blank group,
- N: dilution factor,
- K: slope value of p-nitroaniline standard curve,
- T: reaction time (h),
Product Deacetylation Analysis
Because the free radicals of chitosan are alkaline, acid-base titration is used to determine the degree of deacetylation. During the reaction process, a colloidal solution of chitosan is formed with a fixed amount of acid, and the un-reacted acid can be used to carry out the back-titration with alkali to deduce the amount of chitosan radical-binding acid, and then the amount of the ammonia-free radicals in chitosan can be calculated.
The degree of deacetylation DD% of the product can be calculated by the following formula (2):
Formula 2
- C1: Concentration of standard HCl solution (mol/L),
- V1: Volume of HCl added (mL),
- C2: Concentration of standard NaOH solution (mol/L),
- V2: Volume of NaOH added (mL),
- G: Weight of the sample (g),
- 0.016: Amount of amino acid equivalent to 1mL of 0.1mol/L HCl.
3.2 CsnB Enzyme Performance Test
- CsnB Enzyme Activity Assay
We utilized the 3,5-dinitrosalicylic acid (DNS) method to determine the activity of the CsnB enzyme. The experimental principle of the DNS method for enzyme activity is based on the reaction of reducing sugars with DNS to form colored compounds. First, the enzyme is mixed with a substrate and reacted, and the enzyme catalyzes the formation of reducing sugar from the substrate. At the end of the reaction, a DNS solution is added and heated, and the resulting reducing sugar reacts with DNS to produce a color change. By determining the absorbance value at 540 nm, the concentration of reducing sugar can be reflected, and then the enzyme activity can be deduced. The enzyme activity can be obtained by the following equation (3).
Formula 3
- CsnB-Catalyzed Hydrolysis of Chitosan and Product (Chitooligosaccharides) Analysis
Hydrolysis of chitosan by CsnB produces chitooligosaccharides, the analysis of which involves determining the degree of polymerization and the composition of the resulting product. The mechanism of action of CsnB during enzymatic hydrolysis may be to recognize and break specific glycosidic bonds through the interaction of its active center with the chitosan molecule. Thin-layer chromatography (TLC) is used to measure the degree of Chitooligosaccharides polymerization [5, 6]. And high-performance liquid chromatography (HPLC) is used for quantitative detection of products.
4. Dual Enzyme Synergistic Production of COS
4.1 Efficiency Comparison by Dual Enzyme Stepwise Catalysis and Synergistic Catalysis
To investigate the mechanism of action of CDA and CsnB in the degradation of colloidal chitin, the effect of the two enzymes in stepwise or synergistic catalysis for 10 h was analyzed comparatively with 1% colloidal chitin as the substrate, to obtain the difference in the efficiency of chitooligosaccharides when synthesized by the two catalytic methods.
4.2 Optimization of catalytic conditions with two enzymes
To investigate the activity and stability of dual enzyme catalysis, the differences in catalytic efficiency of the dual enzyme catalytic system at different temperatures, pH values, and dual enzyme addition ratios were set to obtain the optimal reaction conditions.
4.3 Analysis of Enzymatic Digests
The product analysis of dual enzyme synergistic catalysis is carried out by HPLC analysis of the products of the dual enzyme synergistic catalysis reaction at different reaction times to verify whether the inhibitory effect of the products has been eliminated.
References
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